US10059932B2 - Mutant of cellulase-producing microorganism, production method of cellulase and production method of cello-oligosaccharide

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Abstract

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Claims

US10059932B2

Publication number: US10059932B2
Application number: US14/776,679
Authority: US
Inventors: Wataru Ogasawara; Takashi Yamaguchi; Yosuke SHIDA; Yoshiro Ishii; Tatsuki WAKAYAMA; Yoshiro KONDA
Original assignee: Inpex Corp; Nagaoka University of Technology NUC
Current assignee: Inpex Corp; Nagaoka University of Technology NUC
Priority date: 2013-03-15
Filing date: 2014-03-14
Publication date: 2018-08-28
Anticipated expiration: 2034-03-14
Also published as: EP2975113B1; BR112015023537A2; JPWO2014142325A1; EP2975113A4; JP6535280B2; WO2014142325A1; US20160046918A1; DK2975113T3; EP2975113A1; BR112015023537B1

An object is to provide a mutant of a cellulase-producing microorganism which produces a cellulase capable of preferentially producing a cello-oligosaccharide during the selective production of the cello-oligosaccharide through enzymolysis of a cellulosic material in the presence of the cellulase, a method for producing the cellulase and a method for producing a cello-oligosaccharide using the cellulase. The present invention relates to a mutant of a cellulase-producing microorganism, in which cellobiohydrolase and β-glucosidase genes are disrupted.

TECHNICAL FIELD

The present invention relates to a mutant of a cellulase-producing microorganism, a production method thereof, a method for producing a cellulase using the mutant and a method for producing a cello-oligosaccharide using the cellulase.

BACKGROUND ART

A cello-oligosaccharide is an oligosaccharide formed by polymerization of two to six glucose molecules and is a linear oligosaccharide in which two to six glucopyranose units are linked by β-1,4-glycosidic bonds. It is anticipated that cello-oligosaccharides will serve as materials that can be used in the fields of functional foods, energy, feed, chemical engineering products or the like.

In a known method for producing cello-oligosaccharides, decomposition of cellulose as a starting material by cellulases is utilized. As shown in FIG. 1, the mechanism of decomposition of cellulose by cellulases includes (1) reaction where cello-oligosaccharides including cellobiose are produced from cellulose by the effect of cellulose degrading enzymes, (2) reaction where glucose is produced from cellulose and (3) reaction where glucose is produced from cello-oligosaccharides by the effect of a β-glucosidase (NPL 1).

As shown in FIG. 1, there are three types of enzymatic activity for cellulases: endoglucanases which randomly decompose cellulose (sometimes abbreviated to EG below); cellobiohydrolases which release cellobiose from the ends of cellulose chains (sometimes abbreviated to CBH below); and β-glucosidases which decompose cellobiose into two glucose molecules (sometimes abbreviated to BGL below).

In order to increase the productivity of cello-oligosaccharides, it is effective to inhibit the decomposition of cello-oligosaccharides by inhibiting a β-glucosidase contained in cellulases and inhibiting the decomposition of the cello-oligosaccharides into glucose. With respect to methods for inhibiting a β-glucosidase contained in cellulases, various attempts including enzyme fractionation using fractionation by chromatography shown in FIG. 2 have been made.

PTL 1 discloses a method for selectively producing cellobiose from cellulose using cellulases obtained by adsorbing cellulases onto a weak-acid cation exchange resin which has been equilibrated at pH 3.5 to 5.0 to remove a β-glucosidase from the cellulases.

PTL 2 discloses a method for producing cello-oligosaccharides by adsorbing the enzyme components contained in cellulases, except for β-glucosidases, onto cellulose or a cellulose-containing substance in advance and then conducting enzymolysis.

PTL 3 describes a method for separating and removing a β-glucosidase from cellulases characterized by filling a tube or tower reactor with a cellulosic material, letting a cellulase solution that contains a cellobiohydrolase and contains, as impurities, a β-glucosidase continuously pass through the reactor in one direction and thus selectively separating and removing the fraction containing the β-glucosidase.

These conventional methods can reduce the amount of β-glucosidase that contaminates during the production step of cellobiose, and cellobiose can be obtained selectively. However, these methods require a step of adsorbing cellulases onto a solid fraction and a step of separating the solid fraction and thus have problems because the process is complex and costs are high.

In addition, a method for inhibiting the β-glucosidase activity has been developed, and PTL 4 describes a method for producing cellobiose in which δ-gluconolactone or gluconic acid, which is a β-glucosidase inhibitor, is made exist during the decomposition of cellulose by cellulases.

In the method described in PTL 5, glucose oxidase is made exist during the decomposition of cellulose by cellulases and δ-gluconolactone is produced from the produced glucose, and as a result it is attempted to inhibit the β-glucosidase activity.

However, the practical application of these methods has been difficult because of the complexity of the separation of cellobiose as the product from δ-gluconolactone, high price of δ-gluconolactone and the like.

CITATION LIST Patent Literature

PTL 1: JP-A-5-115293
PTL 2: JP-A-2006-204294
PTL 3: JP-A-2006-34206
PTL 4: JP-A-4-75594
PTL 5: JP-A-8-33496

Non Patent Literature

NPL 1: Cellulase, issued from Kodansha Scientific, p. 97-104, 1987

DISCLOSURE OF INVENTION Technical Problem

The invention aims to provide a mutant of a cellulase-producing microorganism which produces a cellulase capable of preferentially producing a cello-oligosaccharide during the selective production of the cello-oligosaccharide through enzymolysis of a cellulosic material in the presence of the cellulase, a method for producing the cellulase and a method for producing a cello-oligosaccharide using the cellulase.

Solution to Problem

The present inventors have prepared strains of a cellulase-producing microorganism in which a main β-glucosidase, main cellobiohydrolase genes and a main endoglucanase gene were knocked out by genetic engineering and found that cello-oligosaccharides can be produced highly selectively from a cellulosic material when the cellulases produced by the strains are used. The present invention has been thus completed.

That is, the invention is as follows.

1. A mutant of a cellulase-producing microorganism, wherein a cellobiohydrolase gene and a β-glucosidase gene are disrupted.

2. The mutant according to the above item 1, wherein the cellobiohydrolase genes are a gene belonging to glycoside hydrolase family (GHF) 6 and a gene belonging to GHF7, and the β-glucosidase gene is a gene belonging to GHF1 or GHF3.

3. The mutant according to the above item 2, wherein the β-glucosidase gene is a gene belonging to GHF3.

4. The mutant according to the above item 1, wherein an endoglucanase gene is further disrupted.

5. The mutant according to the above item 4, wherein the endoglucanase gene is a gene belonging to GHF7.

6. The mutant according to any one of the above items 1 to 5, wherein the cellulase-producing microorganism is a microorganism belonging to Trichoderma.

7. The mutant according to the above item 6, wherein the microorganism belonging to Trichoderma is one selected from the group consisting of Trichoderma reesei, Trichoderma viride, Trichoderma atroviride and Trichoderma longibrachiatum.

8. The mutant according to the above item 7, wherein the microorganism belonging to Trichoderma is Trichoderma reesei.

9. The mutant according to the above item 8, wherein the cellobiohydrolase genes are cbh1 gene and cbh2 gene, and the β-glucosidase gene is bgl1 gene.

10. The mutant according to the

above item

8 or 9, wherein the endoglucanase gene is egl1 gene.

11. A method for producing a cellulase characterized in that a cellulase is produced by culturing the mutant according to any one of the above items 1 to 10.

12. A method for producing a cello-oligosaccharide containing a step of decomposing a cellulosic material using a cellulase produced by the mutant according to any one of the above items 1 to 10.

13. A method for producing a mutant of a cellulase-producing microorganism, containing disrupting a cellobiohydrolase gene and a β-glucosidase gene.

Advantageous Effects of Invention

β-Glucosidase genes and cellobiohydrolase genes have been thought to be essential for the existence of a cellulase-producing microorganism, and it has been thought to be difficult to obtain a cellulase-producing microorganism when these genes are disrupted. In the invention, it was found that a cellulase-producing microorganism can live and grow even when a β-glucosidase gene and a cellobiohydrolase gene have been disrupted, and it was also found that the cellulase that the cellulase-producing microorganism produces can produce a cello-oligosaccharide composed of three or more monosaccharide units highly selectively, efficiently and easily.

Since a main β-glucosidase gene of the mutant cellulase-producing microorganism of the invention has been disrupted, the β-glucosidase activity of the cellulase that the mutant produces is decreased significantly. Also, since a main cellobiohydrolase gene of the mutant has been disrupted, a cello-oligosaccharide composed of three or more monosaccharide units can be produced highly selectively when the cellulase produced by the mutant is used.

In addition to a main β-glucosidase gene and a main cellobiohydrolase gene, a main endoglucanase gene of the mutant has been disrupted, and thus a cello-oligosaccharide composed of three or more monosaccharide units can be produced with higher selectivity.

Therefore, the cellulase produced by the mutant cellulase-producing microorganism of the invention can produce a cello-oligosaccharide highly selectively from a cellulosic material.

Furthermore, when the cellulase produced by the mutant cellulase-producing microorganism of the invention is used, a cello-oligosaccharide can be produced highly selectively from a cellulosic material without any complicated purification step and at a low cost.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic diagram of the mechanism of decomposition of cellulose by cellulases.

FIG. 2 shows a schematic diagram of enzyme fractionation using fractionation by chromatography.

FIG. 3 shows a schematic diagram of a method for producing a mutant which produces an endoglucanase only from a cellulase-producing microorganism by disrupting its genes.

FIG. 4 shows a schematic diagram of the cellulose degrading enzymes produced by Trichoderma reesei.

FIG. 5 shows a schematic diagram of a method for producing a cello-oligosaccharide by disrupting genes of a cellulase-producing microorganism.

FIG. 6 shows schematic diagrams explaining the pyr4 selection marker.

FIG. 7 shows schematic diagrams of marker recycling method using a pyr4-knockout strain.

FIG. 8 shows a schematic diagram of the disruption of egl1 gene using a pyr4-knockout strain.

FIG. 9 shows schematic diagrams of a method for producing a multiple gene-knockout strain using marker recycling method.

FIG. 10 shows a schematic diagram of transformation of Trichoderma reesei.

FIG. 11 shows the results of SDS-PAGE of the transformants produced in the Examples.

Panel (a) of FIG. 12 shows the protein concentrations of the culture solutions of the transformants produced in the Examples. Panels (b) to (d) of FIG. 12 show the results of evaluation of the activities of the enzymes obtained from the transformants produced in the Examples.

FIG. 13 shows the results of evaluation of the activities of the enzymes obtained from the transformants produced in the Examples.

FIG. 14 shows the results of evaluation of the xylanase activities of the enzymes obtained from the transformants produced in the Examples.

FIG. 15 shows the results of evaluation of the oligosaccharide productivity using enzymes obtained from single enzyme-knockout strains.

FIG. 16 shows the results of evaluation of the oligosaccharide productivity using enzymes obtained from a triple enzyme gene-knockout strain (ΔC2ΔC1ΔB1).

FIG. 17 shows the results of evaluation of the oligosaccharide productivity using fractionated enzymes prepared by removing CBHI, CBHII and β-glucosidase 1 from the cellulases obtained from the parent strain and using cellulases obtained from a cbh1-, cbh2- and bgl1-knockout strain (ΔC2ΔC1ΔB1, a triple enzyme gene-knockout strain).

FIG. 18 shows the results of evaluation of the oligosaccharide productivity using enzymes obtained from a quadruple enzyme gene-knockout strain (ΔC2ΔC1ΔB1ΔEG1).

FIG. 19 shows the results of evaluation of the oligosaccharide productivity and the influence of the saccharification period using enzymes obtained from a quadruple enzyme gene-knockout strain.

DESCRIPTION OF EMBODIMENTS

1. Mutant of Cellulase-Producing Microorganism

The invention relates to a mutant of a cellulase-producing microorganism in which a cellobiohydrolase gene, a β-glucosidase gene and an endoglucanase gene have been disrupted. Examples of the cellulase-producing microorganism include microorganisms described in Cellulase (issued from Kodansha Scientific (1987)) and Cellulose No Jiten (issued from Asakura Publishing Co., Ltd. (2000)), such as those of Trichoderma, Acremonium, Aspergillus, Bacillus, Pseudomonas, Penicillium, Aeromonus, Irpex, Sporotrichum and Humicola. Microorganisms belonging to Trichoderma are preferable among the examples.

Examples of microorganisms belonging to Trichoderma include Trichoderma aggressivum, Trichoderma atroviride, Trichoderma asperellum, Trichoderma aureoviride, Trichoderma austrokoningii, Trichoderma brevicompactum, Trichoderma candidum, Trichoderma caribbaeum varaequatoriale, Trichoderma caribbaeum var. Caribbaeum, Trichoderma catoptron, Trichoderma cremeum, Trichoderma ceramicum, Trichoderma cerinum, Trichoderma chlorosporum, Trichoderma chromospermum, Trichoderma cinnamomeum, Trichoderma citrinoviride, Trichoderma crassum, Trichoderma cremeum, Trichoderma dingleyeae, Trichoderma dorotheae, Trichoderma effusum, Trichoderma erinaceum, Trichoderma estonicum, Trichoderma fertile, Trichoderma gelatinosus, Trichoderma ghanense, Trichoderma hamatum, Trichoderma harzianum, Trichoderma helicum, Trichoderma intricatum, Trichoderma konilangbra, Trichoderma koningii, Trichoderma koningiposis, Trichoderma longibrachiatum, Trichoderma longipile, Trichoderma minutisporum, Trichoderma oblongisporum, Trichoderma ovalisporum, Trichoderma petersenii, Trichoderma phyllostahydis, Trichoderma piluliferum, Trichoderma pleuroticola, Trichoderma pleurotum, Trichoderma polysporum, Trichoderma pseudokoningii, Trichoderma pubescens, Trichoderma reesei, Trichoderma rogersonii, Trichoderma rossicum, Trichoderma saturnisporum, Trichoderma sinensis, Trichoderma sinuosum, Trichoderma sp. MA 3642, Trichoderma sp. PPRI 3559, Trichoderma spirale, Trichoderma stramineum, Trichoderma strigosum, Trichoderma stromaticum, Trichoderma surrotundum, Trichoderma taiwanense, Trichoderma thailandcum, Trichoderma thelephorucolum, Trichoderma theobromicola, Trichoderma tomentosum, Trichoderma, Trichoderma, Trichoderma, Trichoderma velutinum, Trichoderma virens, Trichoderma viride and Trichoderma viridescens. From a viewpoint of secretory productivity of cellulases, Trichoderma reesei, Trichoderma viride, Trichoderma atroviride or Trichoderma longibrachiatum is preferable among the examples, and Trichoderma reesei is more preferable.

Example strains of Trichoderma reesei include Trichoderma reesei QM9414 and Trichoderma reesei PC-3-7.

The genes that are disrupted in the invention are a cellobiohydrolase gene and β-glucosidase gene. As shown in FIG. 3, when a cellobiohydrolase gene and a β-glucosidase gene of a cellulase-producing microorganism are disrupted, a mutant which produces an endoglucanase only can be obtained. Complete removal of the enzyme components is possible using the mutant, as compared to enzyme fractionation, and a cello-oligosaccharide can be produced efficiently.

By further disrupting an endoglucanase gene, a cello-oligosaccharide composed of three or more monosaccharide units can be produced with higher selectivity.

The cellobiohydrolase genes are preferably a gene belonging to glycoside hydrolase family [GHF (www.cazy.org/Glycoside-Hydrolases.html)] 6 and a gene belonging to GHF7. The .beta.-glucosidase gene is preferably a gene belonging to GHF1 or GHF3, more preferably a gene belonging to GHF3.

The cellulose degrading enzymes that Trichoderma reesei produces are shown in FIG. 4. As shown in FIG. 4, the cellulose degrading enzymes of Trichoderma reesei include at least two kinds of cellobiohydrolase (CBHI and CBHII), eight kinds of endoglucanase (EGI to EGVIII) and 10 kinds of β-glucosidase (BGLI, BGLII, Cel1b and Cel3b to Cel3h). Among them, five kinds of β-glucosidase (BGLI, Cel3b, Cel3e, Cel3f and Cel3g) are secreted and released to the outside the microorganism.

Among the two kinds of cellobiohydrolase (CBHI and CBHII) of Trichoderma reesei, CBHI (Cel7a) belongs to GHF7 while CBHII (Cel6a) belongs to GHF6. Among the eight kinds of endoglucanase (EGI to EGVIll), EGI (Cel7b) belongs to GHF7, EGII (Cel5a) belongs to GHF5, EGIII (Cell2a) belongs to GHF12, EGIV (Cel61a) belongs to GHF61, EGV (Cel45a) belongs to GHF45, EGVI (Cel74a) belongs to GHF74, EGVII (Cel61b) belongs to GHF61, and EGVIII (Cel5b) belongs to GHF5. Among the 10 kinds of p-glucosidase (BGLI, BGLII, Cell band Cel3b to Cel3h), BGLI (Cel3a) belongs to GHF3, BGLII (Cel7a) belongs to GHF1, Cell b belongs to GHF1, and Cel3b to h belong to GHF3.

When the microorganism belonging to Trichoderma is Trichoderma reesei, it is preferable that the genes shown in Table 1 (cellobiohydrolase and .beta.-glucosidase genes) are disrupted (FIG. 5). Furthermore, an endoglucanase is preferably disrupted. In this regard, the names, numbers, functions and the like of the genes in Table 1 are described based on GenBank and JGI (Trichoderma reesei) (genome.jgi-psf.org/Trire2/Trire2.home.html).

TABLE 1

		Explanation
Gene Name	Gene No.	of Function	Reference	SEQ ID NO

cbh1	E00389	Cellobiohydrolase	1	1
cbh2	M16190	Cellobiohydrolase	2	2
bgl1	U09580	β-Glucosidase	3	3
cel3b	121735	β-Glucosidase	4	4
cel3e	76227	β-Glucosidase	4	5
Estimated bgl	47268	β-Glucosidase	4	6
(cel3f)
Estimated bgl	104797	β-Glucosidase	4	7
(cel3g)
egl1	M15665	Endoglucanase		5	9

Reference 1: JP 1985149387-A/1

Reference 2: Gene 51(1), 43-52 (1987)

Reference 3: Thesis (1993) Mikrobielle Biochemie, Inst. biochem. Technol

Reference 4: Nat Biotechnol. 26, 553-560 (2008)

Reference 5: Homology between cellulase genes of Trichoderma reeseikoron complete nucleotide sequence of the endoglucanase I gene, Penttila, M., Lehtovaara, P., Nevalainen, H., Bhikhabhai, R. and Knowles, J., Gene 45(3), 253-263 (1986)

In the invention, for the purpose of producing a cello-oligosaccharide composed of three or more monosaccharide units highly selectively, three or more of the eight genes are preferably disrupted. Four or more of the genes are more preferably disrupted, and five or more of the genes are further preferably disrupted. More specifically, it is preferable to disrupt at least three of the genes, namely cbh1, cbh2 and bgl1. It is further preferable to disrupt four of the genes, namely cbh1, cbh2, bgl1 and egl1. Mutants in which these genes have been disrupted can be produced according to the method described in Mol. Gen. Genet. 241 (5-6), 523-530 (1993).

The genes described in Table 1 may be, for example, genes which have the respective nucleotide sequences of the genes having deletion, substitution or addition of one or several nucleotides and which have the same functions as those of the respective genes. Moreover, genes which have the same functions as those of the respective genes shown in Table 1 and/or which have nucleotide sequences with a homology of 70% or more, preferably 80% or more, more preferably 90% or more, further preferably 95% or more, particularly preferably 98% or more to the nucleotide sequences of the respective genes in Table 1 are believed to correspond to the genes shown in Table 1, and these genes are also included in the invention as the genes which may be disrupted.

The genes can be inactivated by a method of inserting another DNA fragment into the genes, a method of introducing a mutation into the transcription/translation start sites of the genes and the like, but it is more preferable to physically delete the target genes.

Processes for disrupting a gene or a group of genes include a method of systematically disrupting the target gene(s) shown in Table 1, as well as a method of introducing random deletion or inactivating mutation into the gene(s) and then evaluating the protein productivity and analyzing the gene(s) by suitable methods.

In order to disrupt a target gene, a method by homologous recombination may be used for example. In other words, a target gene in the genome of the parent strain can be interrupted and inactivated through homologous recombination of a part of the target gene, by cloning a DNA fragment containing a part of the target gene using a suitable plasmid vector and transforming the cells of the parent strain with the obtained circular recombinant plasmid.

In another method, a target gene in the genome can be replaced with a disrupted gene fragment by: constructing a mutant target gene to which an inactivation mutation is introduced by nucleotide substitution, nucleotide insertion or the like, a linear DNA fragment which contains the outer regions of the target gene but does not contain the target gene or the like, by a method such as PCR: transforming the cells of the parent strain with the mutant target gene or the linear DNA fragment: and thus inducing homologous recombination by double crossing over at one site in the outer regions of the mutation site introduced to the target gene in the parent strain genome or at one site in the outer regions of the target gene.

In particular, with respect to a method for disrupting a target gene of a cellulase-producing microorganism through homologous recombination, several examples have already been reported (marker recycling method and the like), and the mutant cellulase-producing microorganism of the invention can be obtained according to such a method.

Example methods for deleting or inactivating a gene at random include a method where homologous recombination similar to those of the above methods is induced using a randomly cloned DNA fragment and a method where the parent strain is irradiated with γ ray or the like.

As a more specific example, a method for producing multiple gene- (cbh1-, cbh2- and bgl1-) knockout strains using marker recycling method [Curr. Genet., 48(3), 204-211 (2005)] is explained below, but the method for disrupting genes of the invention is not limited to the following method.

The gene pyr4 (SEQ ID NO:8) is a selection marker gene, and as shown in FIGS. 6 (a) and (b), a pyr4⁺ strain can grow without the addition of uridine to the culture medium while a pyr4⁻ strain (a pyr4-knockout strain) is resistant to 5-fluoroorotic acid (5-FOA) and is an uridine auxotrophic strain.

Marker recycling method using a pyr4-knockout strain is explained referring to FIG. 7. A cassette for recombination in which the target gene has been replaced with pyr4 and which has direct repeats is introduced into an uridine auxotrophic strain (pyr4⁻) obtained by gene disruption by gene targeting using the 5-FOA resistance as an index, and transformants which grow in a culture medium containing no uridine are obtained [FIG. 7 (a)]. From the obtained transformants, a strain having one copy of the disruption cassette inserted at the target site through double crossover is selected by Southern blotting analysis. Then, spores of the obtained strain are applied on a culture medium containing 5-FOA, and a colony which has regained the resistance to 5-FOA after pyr4 has been removed through intramolecular homologous recombination and which thus grows on the culture medium is used as a target gene-knockout strain [FIG. 7 (b)]. The step is repeated to disrupt multiple genes, and the mutant cellulase-producing microorganism of the invention can be obtained. An example explaining the case where the target gene is egl1 gene is shown in FIG. 8.

Various cellulose materials such as Avicel, powder of filter paper, and biomass containing cellulose are used as the carbon sources for the cultivation. Examples of the nitrogen source are polypeptone, ammonium sulfate, broth, corn steep liquor (CSL) and soybean cake.

In addition, a component necessary for producing the target cellulase may be added to the culture medium. Moreover, the addition of a xylan component to the culture medium can increase the production of xylanase.

For the cultivation of the strains, various culture methods such as shake culture, rotary culture, rotary shake culture, static culture and continuous culture may be employed, but shake culture or rotary culture is preferable. The cultivation temperature is generally from 20° C. to 35° C., preferably from 25° C. to 31° C., and the cultivation period is generally from 4 to 10 days, preferably from 4 to 9 days.

The cellulase thus produced by a mutant can be recovered from the extracellular part of the mutant. The cellulase can be measured by analyzing the cell lysate or the supernatant sample of the culture medium directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, which are known in this technical field.

The cellulase produced during the cell cultivation is secreted in the culture medium and purified or separated for example by removing unnecessary components from the cell culture. Examples of the method for purifying the cellulase include affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, ethanol precipitation, reverse-phase HPLC, cation exchange chromatography for example on silica or DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation and gel filtration. One of these methods may be used alone, or a combination of the methods may be used.

2. Method for Producing Cello-Oligosaccharide

The method for producing a cello-oligosaccharide through enzymolysis of cellulose using the cellulase may be a known method and is not particularly restricted. In an example method, a cellulosic material is suspended as a substrate in an aqueous medium, followed by adding the cellulase produced according to the method of the invention, and saccharification is carried out by heating while stirring or shaking the mixture.

The reaction conditions of the method such as the suspending method, the stirring method, the method and the order for adding the cellulase and the substrate and their concentrations may be suitably adjusted so that the yield of the obtained cello-oligosaccharide becomes higher. The pH and the temperature of the reaction solution should be within ranges in which enzymes are not deactivated, and in general, the temperature may be within the range from 5 to 95° C. and the pH may be within the range from 1 to 11 when the reaction is carried out under the normal pressure.

The cellulosic material may be a water-soluble or water-insoluble material and may be derived from a plant or an animal. Examples of the animals and plants producing the cellulosic material include wood, bamboo, wheat straw, rice straw, corncob, cotton, ramie, bagasse, kenaf, beet, ascidians and bacterial cellulose.

In addition to natural cellulosic material produced by the animals and plants, regenerated cellulosic materials obtained by once chemically/physically dissolving or swelling a natural cellulosic material and then regenerating the substance and cellulose derivative-based substances obtained by chemically modifying a cellulosic material may be used. For industrial purposes, the cellulosic material may be any of natural cellulosic materials such as pulp, cellulose powder and crystalline cellulose, regenerated cellulose such as rayon, various regenerated cellulosic materials such as alkaline cellulose and phosphoric acid-swollen cellulose (PSC), and various cellulose derivatives such as carboxymethylcellulose (CMC) sodium salt.

In this regard, however, in order to use the cello-oligosaccharide obtained for medicines, foods or cosmetics, a natural cellulosic material is preferably used. One kind of the cellulosic materials may be used as the starting material, or a mixture of two or more kinds may be used.

The production process of a cello-oligosaccharide through enzymolysis of cellulose using the cellulase may be batch process or continuous process. In order to avoid the product inhibition due to cellobiose during the enzymolysis, it is preferable from the viewpoint of increasing the productivity of the cello-oligosaccharide to keep the cellobiose concentration in the reaction system within a specific range so that the reaction is not influenced by the product inhibition of the cellulase.

Examples of the method for keeping the cellobiose concentration in the reaction system within a specific range are: a method in which produced cellobiose is removed from the reaction system by membrane filtration such as ultrafiltration or reverse osmosis filtration; a method in which an organic porous base material such as activated carbon and dry plant powder of bamboo, wood or the like, an inorganic porous base material such as silicon dioxide or the like is introduced into the reaction system, and cellobiose is adsorbed onto the base material; a method in which a cellulose substrate is immobilized in a column or the like, and a reaction solution containing the cellulase is made pass through the column or the like; and a method in which the cellulase is immobilized on a polymer or the like, and a reaction solution containing cellulose is made pass through the polymer or the like.

The aqueous solution which contains a cello-oligosaccharide as the main component and which is obtained by the enzymolysis can be subjected to purification treatment such as decolorization, desalination or enzyme removal if necessary. The purification method is not particularly restricted as long as it is a known method, but examples include activated carbon treatment, ion exchange resin treatment, chromatography treatment, filtration treatment such as microfiltration, ultrafiltration or reverse osmosis filtration and crystallization treatment. One of the methods may be used alone, or a combination of two or more of the methods may be used.

The aqueous solution containing a cello-oligosaccharide as the main component purified by the method may be used as it is but may be solidified by drying if necessary. The drying method is not particularly restricted as long as it is a known method, but for example, spray drying, freeze-drying, drum drying, thin film drying, shelf drying, pneumatic conveying drying, vacuum drying or the like may be used. One of the methods may be used alone, or a combination of two or more of the methods may be used.

As the medium for the cello-oligosaccharide during the purification and drying treatment, an organic solvent or the like may be used if necessary in addition to water. The organic solvent used here is not particularly restricted, but for example, those which are used in the steps for producing medicines, foods and additives thereof are preferable. Examples are those classified as solvents in Japanese Pharmaceutical Excipients Directory (issued from Yakuji Nippo Limited.), Japanese Pharmacopoeia and Japan's Specifications and Standards for Food Additives (both are issued from Hirokawa Shoten Co.). Water and the organic solvent may be each used alone, or two or more kinds may be used in combination. Also, the cello-oligosaccharide may be once dispersed in one kind of medium and then dispersed in a different medium after removing the first medium.

The form of the cello-oligosaccharide which has passed through the above steps is not particularly restricted, but for example, the cello-oligosaccharide may be used in the form of a solid, a suspension, an emulsion, syrup or a solution at room temperature. Examples of the solid cello-oligosaccharide include powder, granules, pellets, a compact, a laminate or a solid dispersion.

The use of the cello-oligosaccharide is not particularly restricted, but examples include food components, cosmetic components, dye components, flavor components, therapeutic components in medicines, pesticide components, feed components, fertilizer components, culture medium components, reagent components for analysis, additives, intermediate materials and fermentation materials in the fields of foods, cosmetics, medicines, general industrial products or the like.

Among foods, the cello-oligosaccharide produced by the method of the invention is used for example for gel such as jelly, pudding or yogurt; seasoning such as mayonnaise, dressing, sauce, baste, soup or processed vegetable products; retort-pouch foods and chilled foods such as curry, hashed beef, meat sauce, stew or soup; processed livestock products such as hamburger steak, bacon, sausage, salami sausage or ham; marine paste products such as steamed fish paste, hollow bamboo-shaped fish paste cake, fish meat ham/sausage or steamed and fried fish paste; processed wheat foods such as bread, uncooked noodle, dry noodle, macaroni, spaghetti, bread covering of Chinese filled bun, cake mix, premix, white sauce or skins of gyoza dumpling and spring roll; canned foods and bottled foods such as curry, sauce, soup, food boiled down in sweetened soy sauce or jam; snacks and confectionery such as candy, troche, tablet candy, chocolate, biscuit, cookies, rice crackers, Japanese- and Western-style sweets, cake with cream or fruit, snacks, sugar snacks or pudding; seasoned and processed foods such as deep-fried foods, croquette, gyoza dumpling or Chinese filled bun; pastes such as vegetable paste, minced meat, fruit paste or sea food paste; and the like.

In addition, the cello-oligosaccharide is used for example for dairy products such as ice cream, iced milk, ice cream with milk-solids content of 3% or more, whipped cream, condensed milk, butter, yogurt, cheese or white sauce; processed oils and fats such as margarine, fat spread or shortening; and the like. Moreover, the cello-oligosaccharide may be used for beverages including carbonated drink such as cola, fruit drink which is carbonated, contains alcohol or is mixed with a dairy product, fruit juice, or drink or lactic drink containing fruit; coffee; lactic acid/lactic drink such as milk, soymilk, chocolate milk, fruit milk or yogurt; tea drink such as green tea, oolong tea, powdered green tea or black tea; and the like.

The cello-oligosaccharide produced by the method of the invention is expected to have various physiological activities such as activation of intestinal useful flora, for example activation of lactic acid bacteria, lactobacilli and the like, lowering of blood sugar level and blood insulin level, lowering of blood cholesterol, lowering of body fat percentage, promotion of lipid/glucose metabolism, improvement of bowel movement/odor of feces and cariostatic effect. Thus, the cello-oligosaccharide may be used for functional foods, health foods, diet foods and the like as a physiologically active substance, in addition to the general food uses above.

Among general industrial products, the cello-oligosaccharide is used for example for food components, cosmetic components, dye components, flavor components, therapeutic components in medicines, pesticide components, feed components, fertilizer components, culture medium components, reagent components for analysis, additives, intermediate materials, fermentation materials and the like.

The cello-oligosaccharide produced by the method of the invention has a high purity and thus may be used as a material for chemical conversion into various cello-oligosaccharide derivatives.

The invention is explained more specifically based on the Examples and the like, but the invention is not limited by the Examples and the like.

Examples

[Production of Multiple Gene-Knockout Strains using Marker Recycling]

(1) Production of cbh2-Knockout Strain (ΔC2) and bgl1-Knockout Strain (ΔB1)

As shown in FIG. 9 (a), a cbh2-knockout strain was obtained by marker recycling method. An uridine auxotrophic strain (pyr4⁻) obtained through gene disruption using the resistance to 5-FOA as an index was transformed with a cbh2 gene disruption cassette by protoplast-PEG method, and transformants which grew in a culture medium containing no uridine were obtained. From the obtained transformants, strains each having one copy of the disruption cassette inserted at the target site through double crossover were selected by Southern blotting analysis. Then, spores of the obtained strains were applied on a culture medium containing 5-FOA, and colonies which regained the resistance to 5-FOA after pyr4 was removed through intramolecular homologous recombination and which grew on the culture medium were obtained. One of the strains was used as a cbh2-knockout strain (ΔC2). A bgl1-knockout strain (ΔB1) was produced by a similar method.

(2) Production of cbhl- and cbh2-knockout strain (˜C2˜C 1), cbhl-, cbh2- and bgl1-knockout strain (ΔC2ΔC1ΔB1, triple enzyme gene-knockout strain) and cbh1-, cbh2-, bgl1- and egl1-knockout (ΔC2ΔC1ΔB1ΔEG1, quadruple enzyme gene-knockout strain)

As shown in FIG. 9 (b), a cbhl- and cbh2-knockout strain was produced by marker recycling method using the cbh2-knockout strain. In addition, as shown in FIG. 9 (c) and FIG. 10, a cbhl-, cbh2- and bgl1-knockout strain was produced by marker recycling method using the cbhl- and cbh2-knockout strain. Moreover, a cbh1-, cbh2-, bgl1 and egl1-knockout strain was produced by marker recycling method using the cbh1-, cbh2- and bgl1-knockout strain.

[Evaluation of Gene-Knockout Strains]

In order to confirm the expression of cellulases of the obtained gene-knockout strains, 10⁷spores were inoculated in a 300 mL Erlenmeyer flask containing 50 mL of a liquid culture medium for Trichoderma reesei. The composition of the culture medium was as follows.

- 1% Avicel, 0.14% (NH₄)₂SO₄, 0.2% KH₂PO₄, 0.03% CaCl₂·2H₂O, 0.03% MgSO₄·7H₂O, 0.1% Bacto Polypepton, 0.05% Bacto Yeast extract, 0.1% Tween 80, 0.1% trace element and 50 mM tartaric acid buffer (pH 4.0)

The composition of the trace element was as follows.

- 6 mg of H₃BO₃, 26 mg of (NH₄)₆Mo₇O₂₄·4H₂O, 100 mg of FeCl₃·6H₂O, 40 mg of CuSO₄·5H₂O, 8 mg of MnCl₂·4H₂O and 200 mg of ZnCl₂: the volume was adjusted to 100 ml by adding distilled water

After cultivation at 220 rpm at 28° C. for seven days, the culture solutions were centrifuged at 3000 rpm for 15 minutes. The separated supernatants of the culture solutions were filtered through Sartolab (Sartorius Japan K.K.), and the obtained filtrates were used as the enzyme solutions. FIG. 11 shows the results of the SDS-PAGE of 20 μL of the obtained enzyme solutions.

Among the proteins secreted from Trichoderma reesei, the molecular weights of the cellulases encoded by the disrupted genes are 68 kDa (CBHI), 58 kDa (CBHII), 82 kDa (BGLI) and 54 kDa (EGI). As a result of the SDS-PAGE, the lack of the cellulase components encoded by the disrupted genes was confirmed.

Using the obtained enzyme solutions, (1) protein concentration, (2) β-glucosidase activity, (3) endoglucanase activity, (4) cellobiohydrolase activity, (5) cellobiase activity, (6) avicelase activity, (7) CMCase activity and (8) xylanase activity were measured by the following methods. The results are shown in FIG. 12 (a) to (d), FIG. 13 and FIG. 14.

(1) Protein Concentration

Using Bio-Rad Protein Assay (Bio-Rad Laboratories, Inc.), the absorbance at 595 nm was measured, and the protein concentration was determined using bovine γ globulin as the standard substance.

(2) β-Glucosidase Activity

Using cellobiose as the substrate, the glucose concentration was measured by Glucose CII Test Wako (Wako Pure Chemical Industries, Ltd.), and 1 U of β-glucosidase activity was defined as the amount of enzyme that hydrolyzed 1 μmol of cellobiose per minute.

(3) Endoglucanase Activity

Using carboxymethylcellulose as the substrate, the resulting reducing sugar was measured by Somogyi-Nelson method, and 1 U of endoglucanase activity was defined as the amount of enzyme that released 1 μmol (in terms of glucose) of reducing sugar per minute.

(4) Cellobiohydrolase Activity

Using crystalline cellulose as the substrate, the resulting reducing sugar was measured by Somogyi-Nelson method, and 1 U of cellobiohydrolase activity was defined as the amount of enzyme that released 1 μmol (in terms of glucose) of reducing sugar per minute.

(5) Cellobiase Activity

Using cellobiose as the substrate, the glucose concentration was measured by Glucose CII Test Wako (Wako Pure Chemical Industries, Ltd.), and 1 U of cellobiase activity was defined as the amount of enzyme that released 2 μmol of glucose per minute.

(6) Avicelase Activity

Using Avicel as the substrate, the resulting reducing sugar was measured by Somogyi-Nelson method, and 1 U of avicelase activity was defined as the amount of enzyme that released 1 μmol (in terms of glucose) of reducing sugar per minute.

(7) CMCase Activity

Using carboxymethylcellulose as the substrate, the resulting reducing sugar was measured by Somogyi-Nelson method, and 1 U of CMCase activity was defined as the amount of enzyme that released 1 μmol (in terms of glucose) of reducing sugar per minute.

(8) Xylanase Activity

Using xylan as the substrate, resulting xylose was measured using DNS (dinitrosalicylic acid) method, and 1 U of xylanase activity was defined as the amount of enzyme that released 1 μmol (in terms of xylose) of reducing sugar per minute.

[Production of Fractionated Enzymes by Enzyme Fractionation]

By eluting the fraction adsorbed onto DEAE-sepharose, which is an anion exchange resin, under the condition of 50 mM phosphate buffer (pH 6.0) with NaCl, CBH1 was removed from the cellulases obtained from the ΔC2 strain to obtain an enzyme sample. The enzyme sample was made pass through Mono-S, which is a cation exchange resin, under the condition of 50 mM acetic acid buffer (pH 4.0), and the fraction which was not adsorbed was used as the fractionated enzymes from which β-glucosidase 1 was removed (ΔC2-C1-B1).

[Evaluation of Oligosaccharide Productivity]

The oligosaccharide productivity of cellulases was evaluated using phosphoric acid-swollen cellulose as the substrate by HPLC analysis using KS-802, which is a column for sugar analysis.

(1) Evaluation of Oligosaccharide Productivity Using Enzymes Obtained from Single Gene-Knockout Strains

The results of the evaluation of the oligosaccharide productivity of the cellulases obtained from the parent strain (Trichoderma reesei PC-3-7), the bgl1-knockout strain (ΔB1) and the cbh2-knockout strain (ΔC2) are shown in Table 2 and FIG. 15.

TABLE 2

Sugar Recovery Rate [g-sugar/g-substrate]

	PC-3-7	ΔB1	ΔC2

G1	0.41	0.12	0.55
(Glucose)
G2	0.23	0.58	0.11
(Cellobiose)

As shown in Table 2 and FIG. 15, through the oligosaccharide production using the cellulases obtained from the bgl1-knockout strain (ΔB1), cellobiose could be obtained in an amount 2.5 times as high as that of the parent strain.

(2) Evaluation of Oligosaccharide Productivity Using Enzymes Obtained from Triple Enzyme Gene-Knockout Strain

The results of the evaluation of the oligosaccharide productivity of the cellulases obtained from the cbh1-, cbh2- and bgl1-knockout strain (ΔC2ΔC1ΔB1, a triple enzyme gene-knockout strain) are shown in Table 3 and FIG. 16.

TABLE 3

Sugar Recover Rate [g-sugar/g-substrate]

Enzyme · Concen-
tration · [g/L ]	0.01	0.05	0.10

Reaction ·	24	48	24	48	24	48
Period · [hr],
G1 · (Glucose)	0.084	0.12	0.14	0.19	0.18	0.24
G2 · (Cellobiose)	0.19	0.22	0.23	0.24	0.23	0.23
G3 · (Cellotriose)	0.0063	0.0085	0.01	0.012	0.016	0.018
G4 · (Cellotetraose)	—	—	—	—	0.0033	0.0025

As shown in Table 3 and FIG. 16, it was found that the production amounts of cellotriose and cellotetraose as well as that of cellobiose increase through the oligosaccharide production using the cellulases obtained from the cbh1-, cbh2- and bgl1-knockout strain.

(3) Evaluation of Oligosaccharide Productivity Using Enzymes Obtained by Enzyme Fractionation and Enzymes Obtained from Triple Gene-Knockout Strain

The oligosaccharide productivity was evaluated using the fractionated enzymes (ΔC2-C1-B1) prepared by removing CBH1, CBH2 and β-glucosidase 1 from the cellulases obtained from the parent strain (Trichoderma reesei PC3-7) and the cellulases obtained from the cbh1-, cbh2- and bgl1-knockout strain (ΔC2ΔC1ΔB1, a triple enzyme gene-knockout strain). The results are shown in Table 4 and FIG. 17.

TABLE 4

Sugar Recovery Rate [g-sugar/g-substrate]

	ΔC2-C1-B1	ΔC2ΔC1ΔB1

G1	0.15	0.14
(Glucose)
G2	0.24	0.23
(Cellobiose)
G3 (Cellotriose)	0.1	0.1
G4 (Cellotetraose)	0	0

As shown in Table 4 and FIG. 17, it was found that the cellulases obtained from the triple enzyme gene-knockout strain have oligosaccharide productivity comparable with that of the fractionated enzymes and have excellent productivity also with respect to cellotriose, which is a trisaccharide.

(4) Evaluation of Oligosaccharide Productivity Using Enzymes Obtained from Quadruple Enzyme Gene-Knockout Strain

The results of the evaluation of the oligosaccharide productivity of the cellulases obtained from the cbh1-, cbh2-, bgl1- and egl1-knockout strain (ΔC2ΔC1ΔB1ΔEG1, a quadruple enzyme gene-knockout strain) are shown in Table 5 and FIG. 18.


Sugar Recovery Rate[g-sugar/g-substrate]

Enzyme ·
Concentra-
tion · [g/L]	0.01	0.05

Reaction ·	12	24	48	72	12	24	48	72
Period ·
[hr],
G1 ·	0.016	0.027	0.044	0.052	0.052	0.078	0.11	0.15
(Glucose)
G2 ·	0.068	0.11	0.14	0.16	0.15	0.19	0.22	0.24
(Cellobiose)
G3 ·	0.098	0.14	0.18	0.19	0.18	0.18	0.16	0.15
(Cellotriose)
G4 · (Cello-	0.004	0.002	—	—	—	—	0.003	0.003
tetraose)

Reaction ·	12	24	48	72
Period ·
[hr],
G1 ·	0.073	0.11	0.18	0.23
(Glucose)
G2 ·	0.17	0.22	0.25	0.27
(Cellobiose)
G3 ·	0.17	0.16	0.13	0.12
(Cellotriose)
G4 · (Cello-	0.003	0.004	0.004	0.004
tetraose)

As shown in Table 5 and FIG. 18, it was found that the cellulases obtained from the quadruple enzyme gene-knockout strain have oligosaccharide productivity comparable with that of the fractionated enzymes and have excellent productivity also with respect to cellotriose, which is a trisaccharide. It was found that the disruption of egl1 gene has higher effect of producing a cello-oligosaccharide composed of three or more monosaccharide units.

(5) Oligosaccharide Productivity and Influence of Saccharification Period Using Enzymes obtained from Quadruple Enzyme Gene-Knockout Strain

As shown in FIG. 19, the production amount of cellotriose by the cellulases obtained from the cbh1-, cbh2-, bgl1- and egl1-knockout strain (ΔC2ΔC1ΔB1ΔEG1, a quadruple enzyme gene-knockout strain) reached the maximum of 0.19 g/g-substrate when the saccharification period was 72 hours. When saccharification was carried out for 72 hours or longer, the production amount of the trisaccharide hardly changed, but the production amounts of the disaccharide and monosaccharide increased. From the results, it was found that the speed of the trisaccharide production and the speed of the cellotriose decomposition are in equilibrium at the enzyme saccharification period of 72 hours.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skill in the art that various changes and modifications can be made therein without departing from the sprit and scope thereof. This application is based on Japanese patent application No. 2013-053166 filed on Mar. 15, 2013, the entire contents of which are incorporated hereinto by reference. All references cited herein are incorporated in their entirety.

INDUSTRIAL APPLICABILITY

Cello-oligosaccharides are believed to have various physiological activities and are believed to be very useful as materials for functional foods. However, due to the complexity of the production method and the high production cost, cello-oligosaccharides have not been applied to industrial uses, and cellobiose, which is a disaccharide, accounts for 90% or more of the products distributed as cello-oligosaccharides. Moreover, pure cello-oligosaccharides composed of three or more cello-saccharide units are difficult to obtain, and their physiological functions have hardly been analyzed. The mutant cellulase-producing microorganism of the invention can produce not only cellobiose but also an oligosaccharide composed of three or more monosaccharide units and thus is very useful from the industrial and academic viewpoints.

Concentra-

tion · [g/L]

The invention claimed is:

1. A mutant of a cellulase-producing microorganism, wherein two cellobiohydrolase genes, an endoglucanase gene and a β-glucosidase gene are completely physically deleted in the mutant, wherein the cellobiohydrolase genes are cbh1 and cbh2, the endoglucanase gene is egl1, and the β-glucosidase gene is bgl1, wherein no more than two cellobiohydrolase genes, no more than one endoglucanase gene and no more than one β-glucosidase gene are deleted or inactivated in the mutant,

wherein the mutant maintains cellulase-producing activity,

wherein said cellulase produced by the mutant is capable of producing cellotriose from a cellulosic material,

wherein the mutant is a microorganism belonging to the genus Trichoderma, and

wherein the specific activity of carboxymethylcellulase (CMCASE) produced by the mutant is below the specific activity of CMCASE produced by the native parent microorganism when cultured under the same conditions.

2. The mutant according to claim 1, wherein the microorganism belonging to the genus Trichoderma is one selected from the group consisting of Trichoderma reesei, Trichoderma viride, Trichoderma atroviride and Trichoderma longibrachiatum.

3. A method for producing a cellulase comprising culturing the mutant according to claim 1 in a culture medium to produce the cellulase.